Amyloid-like fibrils and aggregates are found widespread in nature. For example, protein aggregates are found as inclusion bodies in bacteria. Such inclusion bodies may in particular arise during the recombinant expression of proteins in bacteria. In addition, a variety of diseases, both in humans and animals, is characterized by the pathogenic formation of amyloid-like fibrils or protein aggregates in neuronal tissues. A well-known and typical example of such diseases is Alzheimer's disease (AD). AD is characterized by the formation of neurofibrillar tangles and β-amyloid fibrils in the brain of AD patients. Similarly, scrapie is associated with the occurrence of scrapie-associated fibrils in brain tissue.
Another class of these diseases is characterized by an expansion of CAG repeats in certain genes. The affected proteins display a corresponding polyglutamine expansion. Said diseases are further characterized by a late onset in life and a dominant pathway of inheritance.
A typical representative of this class of diseases is Huntington's disease. Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by personality changes, motor impairment and subcortical dementia (Harper, 1991). It is associated with a selective neuronal cell death occurring primarily in the cortex and striatum (Vonsattel et al., 1985). The disorder is caused by a CAG/polyglutamine (polygln) repeat expansion in the first exon of a gene encoding a large ˜350 kDa protein of unknown function, designated huntingtin (HDCRG, 1993). The CAG repeat is highly polymorphic and varies from 6-39 repeats on chromosomes of unaffected individuals and 35-180 repeats on HD chromosomes (Rubinsztein et al., 1996; Sathasivam et al., 1997). The majority of adult onset cases have expansions ranging from 40-55 units, whereas expansions of 70 and above invariably cause the juvenile form of the disease. The normal and mutant forms of huntingtin have been shown to be expressed at similar levels in the central nervous system and in peripheral tissues (Trottier et al., 1995a). Within the brain, huntingtin was found predominantly in neurons and was present in cell bodies, dentrites and also in the nerve terminals. Immunohistochemistry, electron microscopy and subcellular fractionations have shown that huntingtin is primarily a cytosolic protein associated with vesicles and/or microtubules, suggesting that it plays a functional role in cytoskeletal anchoring or transport of vesicles (DiFiglia et al., 1995; Gutekunst et al., 1995; Sharp et al., 1995) Huntingtin has also been detected in the nucleus (de Rooij et al., 1996; Hoogeveen et al., 1993) suggesting that transcriptional regulation cannot be ruled out as a possible function of this protein.
In addition to HD, CAG/polygin expansions have been found in at least six other inherited neurodegenerative disorders which include: spinal and bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), and the spinocerebellar ataxias (SCA) types 1, 2, 3 and 6 (referenced in Bates et al. 1997). The normal and expanded size ranges are comparable with the exception of SCA6 in which the expanded alleles are smaller and the mutation is likely to act by a different route. However, in all cases the CAG repeat is located within the coding region and is translated into a stretch of polygin residues. Although the proteins harboring the polygin sequences are unrelated and mostly of unknown function, it is likely that the mutations act through a similar mechanism. Without exception, these proteins are widely expressed and generally localized in the cytoplasm. However, despite overlapping expression patterns in brain, the neuronal cell death is relatively specific and can differ markedly (Ross, 1995), indicating that additional factors are needed to convey the specific patterns of neurodegeneration.
In the art, there is a variety of methods to determine the presence of amyloid-like fibrils or protein aggregates. For example, inclusion bodies in bacteria can be made visible microscopically. Further, amyloid-like fibrils such as from Alzheimer's disease may be analyzed by complex methodology; see, for example, Booth et al. Nature 385 (1997), 787-793, and references cited therein.
So far, however, a simple method that may be established in any laboratory without sophisticated equipment is not available. Such a method would be especially useful in routine procedures such as the testing of patient samples for amyloid-like fibrils or protein aggregates. Accordingly, the technical problem underlying the present invention was to provide a rather simple method that may routinely be used for the detection of such fibrils or aggregates. The solution to said problem is provided by the embodiment characterized in the claims.